With the first development of artificial cells (Chang, T .M. S., Science, 1964, 146:524-525), the microencapsulation of biological materials has become an area of study for industrial processes and biomedical research. In 1965, T. M. S. Chang devised the first method for cell encapsulation (Chang, T. M. S., "semipermeable aqueous microcapsules," 1965, Ph.D. Thesis, McGill University; Chang, T. M. S. et al., Can J. Physiol. Pharmacol., 1966, 44:115-28) and proposed the encapsulation of endocrine cells such as pancreatic or hepatic cells for treating pancreatic and liver disorders (Chang, T. M. S., Artifical Cells, 1972, Springfield, Ill., U.S.A., Charles C. Thomas Publishers). A. M. Sun et al. later demonstrated that intra-peritoneally implanted polylysine-alginate microcapsules containing pancreatic islet cells can temporarily maintain normal blood glucose levels in diabetic rats (Sun A. M. et al., Appl. Biochem. Biotechnol., 1984, 10:87-99). When these encapsulated islets were implanted into different animal species such as mice, there was severe aggregation and clumping of the microcapsules. This caused these islets to stop functioning.
For our transplantation studies, we encapsulated rat hepatocytes within the same alginate-polylysine microcapsules used by Sun, A. M. et al. After implantation in mice, we saw severe aggregation of the microcapsules. As determined by trypan blue stain exclusion, cell viability was only noted in the non-aggregated and free floating microcapsules (Wong, H. and Chang, T. M. S., Biomat Art Cells Art. Org., 1988, 16:731-39).
Our studies also showed cells embedding within the microcapsule membrane matrix and protruding from the surface of the microcapsule membrane. These resulted into two major problems. First, the protruding cells can break away and leave holes in the microcapsule membrane. This lets some encapsulated contents to escape out of the microcapsule. Secondly, the protruding cells that remained embedded in the membrane do not get entirely covered by the microcapsule membrane. Thus, these cells are exposed to the outsides of the microcapsule. In addition, poly-l-lysine is biologically reactive. In normal situation, it is covered by a layer of alginate and rendered unobtrusive. Therefore, around the holes and around the cells protruding from the membrane, some of the reactive poly-l-lysine becomes uncovered. The combined effects of these three problems therefore leads to a cascade of immunological events and bioincompatibility which result in host rejection and premature microcapsule aggregation. Therefore, the method of cell encapsulation using the standard polylysine-alginate procedure is not suitable for use in transplantation.
Further `in vivo` studies showed that most of the standard microcapsules containing rat hepatocytes aggregate into clumps when implanted intra-peritoneally into mice. Thus, progressively with time, a fewer number of free unaggregated microcapsules were recovered by periotoneal lavage. In contrast, in mice implanted with blank alginate-polylysine microcapsules containing no cells, there was observed little to no aggregation of the blank microcapsules. There was a greater degree of incompatibility among the hepatocyte containing microcapsules. In hepatocyte loaded microcapsules, we observed cells entrapped in the membrane matrix, and cells protruding from the membrane surface. This exposes the cells to immunological rejection and also exposing some polylysine with resulting bioincompatibility. The result is the aggregation and rejection of the microcapsules. Therefore, cells microencapsulated by the standard polylysine-alginate method cannot function after implantation.
The Japanese Patent Application S.N. 58/189,031, published on Nov. 4, 1983, discloses a method to obtain double microcapsules, which a very similar method was already published by T. M. S. Chang (Chang, T. M. S., 1965, Ph.D. Thesis, `ibid`; Chang, T. M. S. et al., Can J. Physiol. Pharmacol., 1966, `ibid`; Chang, T. M. S., Artifical Cells, 1972, `ibid`).
There is disclosed large microcapsules each containing a number of smaller microcapsules. The smaller microcapsules remain as permanent microcapsules made of permanent outer polymer membranes.
The resulting large microcapsules, although devoided of any encapsulated material embedded within their membrane, have the following three barriers to permeability for the diffusion of substances into and out of the smaller microcapsules:
1) the membrane of the larger microcapsule; PA1 2) the membrane of the smaller microcapsules located within the large one; and PA1 3) the space between the membrane of the larger microcapsule and the membrane of the smaller microcapsules. PA1 1) the exchange of the nutrients required for the growth of the encapsulated cells; PA1 2) the removal of the waste products of the encapsulated cells; PA1 3) the response of the encapsulated cells will be delayed. For example, the response of encapsulated islets of Langerhams in secreting insulin, according to the change in blood glucose concentration, will be delayed and out of sequence; and PA1 4) the low permeability which causes other problems. PA1 a) suspending said small gelled beads in a solution of a water-soluble substance which can be reversibly gelled; PA1 b) forming large droplets from the suspension of step a), thereby obtaining large droplets which contain one or more small gelled beads; PA1 c) gelling said large droplets to produce discrete shape-retaining temporary large gelled beads which contain said small gelled beads; PA1 d) forming semi-permeable membranes on each large gelled beads of c), thereby preventing the incorporation of any biologically active material of the smaller gelled beads into said membranes; and PA1 e) reliquifying the gel within said membranes, thereby dipersing the content of said smaller gelled beads within said capsule.
In microcapsulation of cells, these permeability barriers will adversely affect the following:
Multiple coating and multiple membrane layers around each microcapsule can also prevent cells from embedding in the outer membrane. However, these multiple membranes layer would have problems similar to the double microcapsules. Each layer contributing to the increase in barrier to permeability. The more there is membrane layers, the less permeability is observed.
It would be highly desirable if there could be provided a novel method of microencapsulation of biological materials which would overcome all the problems of the standard methods.
That is, if there could be provided a microcapsule where no cells or biological materials is seen protruding nor entrapped within the capsule membrane matrix and where its permeability is not hampered.
Also, it would also be highly desirable to have a microcapsule which does not tend to aggregate after its implantation.